1. Field of the Invention
The present invention relates generally to the field of Electrophoresis process involving the visualization, documentation and analysis of DNA and RNA gels. The invention specifically involves the photographic process and the analysis capability of the process.
2. Description of the Prior Art
Agarose Gel of DNA
The standard method used to separate, identify, and purify DNA fragments is electrophoresis though agarose gels. This technique is rapid, simple and is able to resolve mixtures of DNA fragments that can not be resolved with other methods. Moreover, the location of the DNA within the gel can be directly determined. DNA bands are stained with ethidium bromide for visualization. Ethidium bromide is a fluorescent dye substance containing a planar group able to intercalate between the stacked bases of the DNA. UV-irradiation absorbed by the DNA at 260 nm and transmitted to the dye, or irradiation absorbed at 300 nm and 360 nm by the bound dye itself, is emitted at 590 nm in the red-orange region of the visible spectrum. This dye can be used to detect both single- or double-stranded DNA. Strands of DNA larger than lkb are separated on agarose gels. Alkaline agarose gels are used to analyze the size of the DNA strand in DNA RNA hybrids that are nuclease-S1-resistant. They are also used to check the size of the first and second DNA stands synthesized by reverse transcriptase. DNA analyzed by alkaline gel electrophoresis is labeled with .sup.32 P, which can be detected with autoradiography. Polyacrylamide Gel electrophoresis is used to analyze and prepare fragments of DNA less than 1 kb in length. These gels are poured between two glass plates that are held apart by spacers to shield the acrylamide solution from exposure to air. This allows for the inhibition of polymerization by the oxygen to be restricted only to a narrow area at the top of the gel. The DNA embedded in polyacrylamide gels can be stained with ethidium bromide where the gel is submerged in staining solution containing ethidium bromide and thus can then be viewed and photographed under UV light. It can also be viewed using autoradiography where resulting bands are viewed and photographed under white light illumination. Strand separation of small DNA fragments, less than 200 nucleotide long, are separated using .sup.32 P labeled DNA fragments and visualized using radiography techniques.
Gel Electrophoresis of RNA
There are two systems frequently used to measure the molecular weight of RNA and to separate RNA's of different sizes for different uses such as Northern blots or in-vitro translation. The first is agarose gel electrophoresis after denaturation of the RNA with glyoxal and dimethylsulfoxide. The second way is electrophoresis of agarose gels that contain methylmercuric hydroxide or formaldehyde. In each case, the RNA is fully denatured, and its rate of migration through the gel is in linear proportion to the log.sub.10 of its molecular weight. Ethidium bromide is used to detect single- and double-stranded RNA.
Methylene Blue
Methylene blue is another method to stain and visualize both DNA and RNA in gels. Separated bands appear blue in color when exposed to white light. It is used to stain DNA and RNA on agarose gels electrophoresis and for DNA fingerprinting visualization.
Autoradiography
Radioactive nucleic acids can be detected by autoradiography, for example for DNA sequence determination, Southern, Western, and Northern blots. Autoradiography is a sensitive method which gives a higher resolution, and does not involve destruction of the sample. The isotope .sup.32 P is mostly used but other weaker .beta.-emitting isotopes are also used for these purposes. The radioactively labeled nucleic acids are exposed for a period of several hours to several days onto X-ray film. The x-ray negatives developed are then visualized and/or photographed to analyzed and sequence determine the resulting nucleic acid band patterns under a uniform source of white light. In the cases of blots, Electrophoresis and staining of Proteins
Fractionation of proteins in polyacrylamide gels is one of the primary means of their characterization due to its speed and ease of use. Many methods to separate both native (undenatured) and denatured proteins exists in the scientific literature. The most widely used technique, however, remains to be SDS polyacrylamide denaturing gel electrophoresis (SDS-PAGE). The goal of SDS-PAGE is to separate proteins based on molecular weight. SDS coats the proteins with negative charges so that during electrophoresis, the protein species will migrate towards to the cation side. The speed of migration depends upon the size of the protein. Lighter species will migrate faster thus will be further along on the gel than higher molecular weight proteins. The primary uses of SDS-PAGE are the determination of the size of a protein component, the estimation of protein purity in a solution, the purification of a protein species of other procedures, and the fractionation of a complex protein mixture prior to immunoblotting (Western blots). Two formats of gels are often used, mini-gels and large gels. Mini-gels are used generally for analytical and some preparative fractionation techniques, whereas the latter is used mainly for analytical separation and isolation of large amounts of denatured proteins. Detection of protein species in gels (visible bands) is usually performed post-electrophoretically using a number of dyes and are visualized and photographed under white light. However band resolution is better with less complex protein samples. Coomassie blue is the most general method used for quantifying 1- and 2-D gels with a densitometer. The staining process can take from 3 to 18 hours and can detect 40-50 ng of protein per band. This method is also used to determine if sufficient peptide is present on a membrane for sequencing. Fast Coomassie Stain is compatible with all Coomassie blue applications. It is a quick alternative to Coomassie blue method but band intensities are slightly less than with standard Coomassie stains. However, it provides a clear background. It is performed in 90 minutes and can detect 50 ng of proteins. Ghostband stains allow for fast and reversible negative staining of proteins, however these stains are not generally recommended for protein recovery. It a 15 minute method that can detect as little as 10-25 ng of protein per band. Silver stains are more sensitive than Coomassie blue detecting as little as 1-5 ng of protein per band. It is however, not recommended for quantification due to protein-to-protein variation and non-linearity of response. Other methods are used to detect the fractionate protein species besides staining. Many of these techniques rely on the transfer of SDS-PAGE-separated proteins to a membrane, others can be detected from the gel. Some of these techniques involve labeling proteins with .sup.35 S, .sup.3 H, .sup.14 C, and .sup.125 I. In such cases, the results are capture on X-ray film and one is able to use autoradiography to visualize the fractionated protein species. One method is using immunoblotting techniques including Western blots and dot blots, in which the sample to be examined is immobilized on a membrane prior to detection. Another common immunoblotting technique is colony/plaque lifts which uses similar procedures for detection. Colony/plaque lifts are used for cDNA expression libraries for clone identification. Western and dot blots are used in screening protein expression, including developmental changes, tissue-to-tissue differences, and in vitro translation reactions. They are also used in monitoring purification of proteins and screening monoclonals. Determining epitopes, however, is only performed using Westerns. Western blots, dot blots, and colony/plaque lifts all require the transfer, or immobilization, of a sample onto a membrane. This is the step where all three methods differ. In western blots, proteins are transferred to a membrane after PAGE; for dot blots non-denatured antigen-containing samples are spotted directly onto a membrane using a pipette. In colony/plaque lifts, intact colonies or plaques are transferred to a membrane, then lysed to expose and bind the potential antigen. Detection of blots and lifts are done through calorimetric reaction, chemiluminescence, and autoradiography.
Prior U. S. Letters Patent which relate to the field of this present invention are as follows:
1. U.S. Pat. No. 3,525,864 by Leach describes a process of removing the luminescent material from the inner surface of the gas discharge tube and instead coating the surface above the tube. The present invention utilizes the tubes with coating in place and uses the fluorescent dyed polymer material to create the required visible light.
2. U.S. Pat. No. 4,059,767 by Macovski describes an apparatus for enhancing the contrast of a transparency. It does not use ultraviolet light and is for transparencies only.
3. U.S. Pat. No. 5,554,449 by Tonomura, Matsui, and Morisyhta describes a high luminance thin film device that exhibits x-ray diffraction patterns. This device is not applicable to visible light emission.